Hans Rasmussen

Experimental evidence against the mitochondrial theory of aging A study of isolated human skeletal muscle mitochondria

The mitochondrial theory of aging was tested with optimised preparation techniques. Mitochondria were isolated from approximately 90 mg quadriceps muscle from healthy humans at age 70+ and 20+. The content of mitochondrial protein was approximately 10 mg g(-1) muscle and the yields were approximately 40%. The mitochondrial integrity was high as judged from the respiratory control and P/O ratios. No general membrane alterations or changes in the cytochrome contents were observed. BSA decreased the non-phosphorylating rates of respiration equally in both age groups. Thirteen different enzyme activities were assayed and normalised to protein content and citrate synthase activity. Most of the critical levels for detection of declines were <10%. In the 70+ group, the activity for fatty acid oxidation was decreased by approximately 20%. Two inherently low activities associated with oxidation of sarcoplasmic NADH were also decreased, probably related to the age change of fibre types. The remaining activities measured, e.g. those of pyruvate dehydrogenase, tricarboxylic acid cycle, respiratory chain, and ATP synthesis, were not observed to be lowered. Thus, the central bioenergetic systems appeared unaltered with age. The obvious discord with reported age declines of human skeletal muscle mitochondrial function is discussed. It is concluded that the present results are incompatible with the mitochondrial theory of aging.

Decreased Frontal Serotonin2A Receptor Binding in Antipsychotic-Naive Patients With First-Episode Schizophrenia

CONTEXT Postmortem investigations and the receptor affinity profile of atypical antipsychotics have implicated the participation of serotonin(2A) receptors in the pathophysiology of schizophrenia. Most postmortem studies point toward lower cortical serotonin(2A) binding in schizophrenic patients. However, in vivo studies of serotonin(2A) binding report conflicting results, presumably because sample sizes have been small or because schizophrenic patients who were not antipsychotic-naive were included. Furthermore, the relationships between serotonin(2A) binding, psychopathology, and central neurocognitive deficits in schizophrenia are unclear. OBJECTIVES To assess in vivo brain serotonin(2A) binding potentials in a large sample of antipsychotic-naive schizophrenic patients and matched healthy controls, and to examine possible associations with psychopathology, memory, attention, and executive functions. DESIGN Case-control study. SETTING University hospital, Denmark. PARTICIPANTS A sample of 30 first-episode, antipsychotic-naive schizophrenic patients, 23 males and 7 females, and 30 matched healthy control subjects. INTERVENTIONS Positron emission tomography with the serotonin(2A)-specific radioligand fluorine 18-labeled altanserin and administration of a neuropsychological test battery. MAIN OUTCOME MEASURES Binding potential of specific tracer binding, scores on the Positive and Negative Syndrome Scale, and results of neuropsychological testing. RESULTS Schizophrenic patients had significantly lower serotonin(2A) binding in the frontal cortex than did control subjects. A significant negative correlation was observed between frontal cortical serotonin(2A) binding and positive psychotic symptoms in the male patients. No correlations were found between cognitive functions and serotonin(2A) binding. CONCLUSION The results suggest that frontal cortical serotonin(2A) receptors are involved in the pathophysiology of schizophrenia. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00207064.

Frontal Dopamine D2/3 Receptor Binding in Drug-Naive First-Episode Schizophrenic Patients Correlates with Positive Psychotic Symptoms and Gender

BACKGROUND The aim of the study was to examine extrastriatal dopamine D(2/3) receptor binding and psychopathology in schizophrenic patients, and to relate binding potential (BP) values to psychopathology. METHODS Twenty-five drug-naive schizophrenic patients and 20 healthy controls were examined with single-photon emission computerized tomography (SPECT) using the D(2/3)-receptor ligand [123I]epidepride. RESULTS In the hitherto largest study on extrastriatal D(2/3) receptors we detected a significant correlation between frontal D(2/3) BP values and positive schizophrenic symptoms in the larger group of male schizophrenic patients, higher frontal BP values in male (n = 17) compared to female (n = 8) patients, and - in accordance with this - significantly fewer positive schizophrenic symptoms in the female patients. No significant differences in BP values were observed between patients and controls; the patients, however, had significantly higher BP in the right compared to the left thalamus, whereas no significant hemispheric imbalances were observed in the healthy subjects. CONCLUSIONS The present data are the first to confirm a significant correlation between frontal D(2/3) receptor BP values and positive symptoms in male schizophrenic patients. They are in agreement with the hypothesis that frontal D(2/3) receptor activity is significant for positive psychotic symptoms. Additionally, the data support a thalamic hemispheric imbalance in schizophrenia.

Lactate dehydrogenase is not a mitochondrial enzyme in human and mouse vastus lateralis muscle.

The presence of lactate dehydrogenase in skeletal muscle mitochondria was investigated to clarify whether lactate is a possible substrate for mitochondrial respiration. Mitochondria were prepared from 100 mg samples of human and mouse vastus lateralis muscle. All fractions from the preparation procedure were assayed for marker enzymes and lactate dehydrogenase (LDH). The mitochondrial fraction contained no LDH activity (detection limit ∼0.05 % of the tissue activity) and the distribution of LDH activity among the fractions paralleled that of pyruvate kinase, i.e. LDH was fractionated as a cytoplasmic enzyme. Respiratory experiments with the mitochondrial fraction also indicated the absence of LDH. Lactate did not cause respiration, nor did it affect the respiration of pyruvate + malate. The major part of the native cytochrome c was retained in the isolated mitochondria, which, furthermore, showed high specific rates of state 3 respiration. This excluded artificial loss from the mitochondria of all activity of a possible LDH. It was concluded that skeletal muscle mitochondria are devoid of LDH and unable to metabolize lactate.

Human quadricepts muscle mitochondria: A functional characterization

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in µmol O min-1 g protein-1 (25°C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 μmol O min-1 g protein-1). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 μmol ATP min-1 g protein-1). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.

Serotonin 2A receptor antagonists for treatment of schizophrenia

Introduction: All approved antipsychotic drugs share an affinity for the dopamine 2 (D2) receptor; however, these drugs only partially ameliorate the symptoms of schizophrenia. It is, therefore, of paramount importance to identify new treatment strategies for schizophrenia. Areas covered: Preclinical, clinical and post-mortem studies of the serotonin 5-HT2A system in schizophrenia are reviewed. The implications of a combined D2 and 5-HT2A receptor blockade, which is obtained by several current antipsychotic drugs, are discussed, and the rationale for the development of more selective 5-HT2A receptor antagonists is evaluated. Moreover, the investigational pipeline of major pharmaceutical companies is examined and an Internet search conducted to identify other pharmaceutical companies investigating 5-HT2A receptor antagonists for the treatment of schizophrenia. Expert opinion: 5-HT2A receptor antagonists appear to assume an intermediate position by being marginally superior to placebo but inferior to conventional antipsychotic drugs. Three previous 5-HT2A receptor antagonists have been discontinued after Phase II or III trials, and available Phase IIa data on the remaining 5-HT2A receptor antagonist will need substantial additional validation to be approved as a new treatment strategy against schizophrenia.

Cortical and subcortical 5-HT2A receptor binding in neuroleptic-naive first-episode schizophrenic patients.

The serotonin 5-HT2A receptor is suspected to be involved in a number of psychiatric disorders, including schizophrenia. In particular, atypical antipsychotics have antagonistic effects on the 5-HT2A receptors, supporting a specific role of the 5-HT2A receptor in the pathophysiology of this disease. The aim of this study is to investigate cortical and subcortical 5-HT2A binding in neuroleptic-naive schizophrenic patients. Fifteen neuroleptic-naive patients diagnosed with schizophrenia (age 27.5±4.5 years), 11 men and 4 women, and 15 healthy control subjects matched for age (28.5±5.7 years) and gender underwent a 40 min positron emission tomography (PET) study using the 5-HT2A antagonist, [18F]altanserin, as a radioligand. PET images were co-registered to 3 T magnetic resonance images (MRIs) for each individual subject, and ROIs were applied automatically onto the individual MRIs and PET images. The cerebellum was used as a reference region. The binding potential of specific tracer binding (BPp) was used as the outcome measure. No significant difference was seen in cortical receptor distribution between patients and controls. An increase in 5-HT2A receptor binding in the caudate nucleus was detected in the group of schizophrenic patients (0.7±0.1) when compared to the healthy controls (0.5±0.3) (p=0.02). Our results confirm other in vivo findings of no difference in cortical 5-HT2A receptor binding between first-episode antipsychotic-naive schizophrenic patients and age- and gender-matched healthy control subjects. However, a preliminary finding of increased 5-HT2A binding in the caudate nucleus requires further investigation to explore the relation of subcortical and cortical 5-HT2A receptor binding.

Rat pancreas secretes particulate ecto-nucleotidase CD39

In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar‐ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT‐PCR and Western blotting we show that pancreas expresses the full length ecto‐nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini and intracellularly. In small intercalated/ interlobular ducts, CD39 immunofluorescence was localized on the luminal membranes, while in larger ducts it was localized on the basolateral membranes. Upon stimulation with cholecystokinin‐octapeptide‐8 (CCK‐8), acinar CD39 relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK‐8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti‐CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic juice. This CD39 was confined only to the particulate and not to the soluble fraction of CCK‐8‐stimulated secretion. No CD39 activity was detected in secretion stimulated by secretin. The role of secreted particulate, possibly microsomal, CD39 would be to regulate intraluminal ATP concentrations within the ductal tree. In conclusion, we show a novel inducible release of full length particulate CD39, and propose its role in the physiological context of pancreatic secretion.

P50 Suppression and Its Neural Generators in Antipsychotic-Naïve First-Episode Schizophrenia Before and After 6 Months of Quetiapine Treatment

BACKGROUND Numerous studies have demonstrated sensory gating deficits in schizophrenia. However, only a few longitudinal studies report on the effects of antipsychotic treatment on sensory gating deficits and their results are inconsistent. In the present study, P50 suppression and its neural generators were investigated in antipsychotic-naïve first-episode patients with schizophrenia before and after 6 months of treatment with quetiapine. METHODS Thirty-four antipsychotic-naïve first-episode schizophrenia patients and age and gender matched healthy controls were tested in an auditory sensory gating paradigm at baseline and after 6 months. During this period, the patients were treated with quetiapine, while controls received no treatment. Sixteen patients completed the study. RESULTS Patients showed significant reduced P50 suppression compared with controls at baseline but not at follow-up. Furthermore, a significant positive correlation between baseline P50 suppression and dose of quetiapine at follow-up was found. P50 suppression in patients receiving above median dosages of quetiapine increased significantly from baseline to follow-up. At baseline, a frontocentral source was significantly more active in patients than in controls at the time of the testing stimulus. CONCLUSIONS The present findings suggest that P50 suppression deficits are already present at an early stage of schizophrenia. Furthermore, particularly those patients with more severe gating deficits appeared to need higher dosages of quetiapine, although their clinical symptoms did not seem to indicate this. Quetiapine treatment significantly improved these gating deficits. Furthermore, a frontocentral source in the brain appeared to be involved in the deficient P50 gating of the patients.

SMALL SCALE PREPARATION OF SKELETAL MUSCLE MITOCHONDRIA, CRITERIA OF INTEGRITY, AND ASSAYS WITH REFERENCE TO TISSUE FUNCTION

Mitochondria prepared in small scale from skeletal muscle were studied with respiration measurements and low temperature spectroscopy. The method of preparation was developed for 25–100 mg tissue with pigeon breast muscle as model organ. The yield was 40%. Data collected during the developmental work were used to evaluate criteria of mitochondrial quality. The cytochrome c conservation, i.e. cytochrome c per mitochondrial quantity in the preparation relative to that in the tissue, is a most useful test parameter. It is bounded between 0-100%. Proportionality between the state 3 rate and the cytochrome c conservation was not rejected by statistical tests. The respiratory control ratio (RCR) was also highly correlated to the cytochrome c conservation. These correlations might be extrapolated to 100% conservation to give hypothetical tissue values. The cause for the correlations is discussed. The P/O ratio showed only weak dependence on the cytochrome c conservation and the state 4 rate showed no dependence. Other, rather insensitive test parameters are also discussed. The pigeon breast muscle mitochondria isolated by the final method showed cytochrome c conservation of 73 ± 9% (n = 16). They are compared with pig biceps femoris mitochondria prepared by the same method. The two types of mitochondria show many similarities. Some differences may be explained by a different amount of inner mitochondrial membrane relative to mitochondrial protein. The pig tissue contains ten times less mitochondrial protein than the pigeon tissue does. (Mol Cell Biochem 174: 55–60, 1997)